1. Field of the Invention
The present invention relates to methods of producing polypeptides in respiratory-deficient mutant cells. The present invention also relates to methods for disrupting a gene in a respiratory-deficient mutant cell. The invention further relates to respiratory-deficient mutant cells and methods for obtaining such mutant cells.
2. Description of the Related Art
There are a variety of methods for selecting and maintaining a recombinant DNA molecule in a host cell. One method involves complementation of an auxotrophic mutation with a cloned wild-type gene. However, maintenance of complementing DNA in complex media is difficult where, for example, an amino acid or a nucleic acid may be present, which also relieves the auxotrophy. Another method employs marker genes that can be selected in wild-type recipients, particularly, resistance marker genes such as antibiotic resistance genes. The use of antibiotic resistance genes generally requires an expensive antibiotic in the culture medium and its subsequent removal from the desired product. A further method involves the use of a lethal chromosomal marker which is repressed by a gene borne by a recombinant DNA cloning vector. This method has been successfully applied to bacteria, but is difficult to apply to other hosts, e.g., fungi. Furthermore, the method requires the use of a plasmid-borne repressor gene that does not interfere with the transcriptional activating sequence driving expression of the recombinant DNA molecule.
The instability of an expression vector containing a recombinant DNA molecule in a host cell poses a serious problem for the consistent high-level expression of the recombinant DNA molecule. Upon subculturing of the transformant, the yield of a product encoded by the recombinant DNA molecule may decline dramatically and unpredictably. This is particularly true if expression of the recombinant DNA molecule imparts a deleterious effect on the host cell, e.g., toxicity of the expressed product toward the cell. It is desirable that a microbial culture of the transformant containing the recombinant DNA molecule be selected and maintained so that substantially all of the microbial cells in the culture will contain the recombinant DNA molecule.
There is a particular need in the art for alternative selection and maintenance systems that insure both high level expression and genetic stability of a transformant during cultivation.
U.S. Pat. No. 4,902,620 discloses the introduction of a heme biosynthetic enzyme (5-aminolevulinic acid synthase) gene into a heme-deficient cell for the production of 5-aminolevulinic acid.
It is an object of the present invention to provide new methods for selecting and maintaining recombinant cells in the heterologous expression of genes.